EP3699288A1 - Procédé pour la production in vivo d'arn dans une cellule hôte - Google Patents

Procédé pour la production in vivo d'arn dans une cellule hôte Download PDF

Info

Publication number
EP3699288A1
EP3699288A1 EP19216087.7A EP19216087A EP3699288A1 EP 3699288 A1 EP3699288 A1 EP 3699288A1 EP 19216087 A EP19216087 A EP 19216087A EP 3699288 A1 EP3699288 A1 EP 3699288A1
Authority
EP
European Patent Office
Prior art keywords
rna
target rna
host cell
rnase inhibitor
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19216087.7A
Other languages
German (de)
English (en)
Inventor
Andreas Schmid
Isabel STROBEL
Fabian Johannes EBER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Curevac SE
Original Assignee
Curevac AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curevac AG filed Critical Curevac AG
Priority to EP19216087.7A priority Critical patent/EP3699288A1/fr
Publication of EP3699288A1 publication Critical patent/EP3699288A1/fr
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes

Definitions

  • the present invention is directed to processes for the in vivo production of a target RNA in host cells.
  • the present invention is also directed to vectors comprising a DNA sequence encoding a target RNA and a DNA sequence encoding an RNase inhibitor, and host cells comprising the vectors.
  • the present invention is further directed to the use of host cells and vectors for the in vivo transcription of a DNA sequence in processes for the production of the target RNA.
  • the human genome comprises about 40.000 genes encoded by DNA. This genetic information is transcribed in RNA and then expressed into functional proteins.
  • Recombinant DNA as well as recombinant RNA have found use for the therapeutic administration in the context of immunotherapy, gene therapy or genetic vaccination.
  • Immunotherapy, gene therapy and genetic vaccination belong to the most promising and quickly developing methods of modern medicine. They may provide highly specific and individual treatment options for therapy of a large variety of diseases. Particularly, inherited genetic diseases, but also autoimmune diseases, infectious diseases, cancerous or tumor-related diseases as well as inflammatory diseases may be the subject of such treatment approaches. It is also envisaged to prevent (early) onset of such diseases by these approaches.
  • RNA does not need to cross the nuclear barrier for its encoded proteins to be expressed (reviewed in Tavernier et al., J Control Release. (2011); 150(3):238-47 .
  • WO 02/24904 A2 discloses the production of RNA in microorganisms in which the RNA formed after transcription is to be secreted into the extracellular medium.
  • the general feasibility of this approach is questionable, since the vector construct used in this method lacked a terminator signal and the ability of microorganisms to secrete large amounts of recombinant RNA is also doubtful.
  • the present invention is directed to a process for producing a target RNA in a host cell comprising the steps of:
  • the host cell is a bacterial cell and more preferably it is E.coli.
  • the target RNA is selected from non-modified RNA and modified RNA, wherein the modified RNA comprises at least one modified nucleotide.
  • the target RNA is selected from the group consisting of mRNA, viral RNA, retroviral RNA and replicon RNA, bicistronic or multicistronic RNA, small interfering RNA (siRNA), antisense RNA, CRISPR RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).
  • the target RNA does not comprise a secondary structure of transfer RNA (tRNA).
  • the target RNA has a length of at least 100 or 200 nucleotides, more preferably of at least 300 or 400 nucleotides, even more preferably of at least 500 or 600 nucleotides and most preferably of at least 700 or 800 nucleotides.
  • the target RNA has a length of not more than 10,000 nucleotides.
  • the target RNA is an mRNA and codes for at least one antigen, a therapeutic protein, an antibody, a B cell receptor, a T cell receptor, or a fragment, variant or derivative thereof.
  • the antigen is a tumor antigen, a pathogenic antigen, an allergenic antigen, or an autoimmune antigen.
  • the host cell additionally comprises a DNA sequence encoding an RNase inhibitor.
  • the DNA sequence encoding an RNase inhibitor can be located on the same vector as the DNA encoding the target RNA or it can be located on a second vector.
  • the DNA sequence encoding an RNase inhibitor may also be integrated into the genome of said host cell.
  • the RNase inhibitor is selected from E.coli RNase inhibitor RraA and human RNase inhibitor RNH1.
  • a compound capable of inhibiting peptide and/or protein synthesis is added in step b). More preferably, the compound capable of inhibiting peptide and/or protein synthesis is chloramphenicol.
  • a substance capable of inhibiting endogenous RNA polymerase is added in step b). More preferably, the substance capable of inhibiting endogenous RNA polymerase added in step b) is a rifamycin which is even more preferred rifampicin.
  • the target RNA is obtained from the host cell by separating the target RNA from the endogenous RNA of the host cell.
  • the step of obtaining the target RNA comprises a step of depleting the ribosomal RNA of the host cell and more preferably the ribosomal RNA of the host cell is depleted by capture hybridization of the ribosomal RNA with complementary oligonucleotides immobilized on a solid phase.
  • the target RNA is obtained by hybridization with a complementary nucleic acid sequence.
  • the complementary nucleic acid sequence is immobilized on a solid matrix.
  • the target RNA comprises an affinity tag capable of binding to an affinity matrix.
  • the affinity tag comprises an aptamer, more preferably the aptamer is capable of binding to a structure useful for purification and even more preferably, the aptamer is capable of binding to Sephadex.
  • the affinity tag may also comprise an RNA nucleotide sequence capable of binding to a protein and/or peptide such asa boxB RNA binding peptide.
  • the target RNA may further comprise a ribozyme sequence, preferably an activatable ribozyme sequence, which is capable of cleaving the target RNA to delete the affinity tag.
  • the ribozyme sequence is preferably located between the RNA to be produced and the affinity tag.
  • the target RNA is obtained by binding of the affinity tag to an affinity matrix.
  • the affinity tag is cleaved from the target RNA by activating the activatable ribozyme sequence.
  • the present invention is further directed to a vector comprising a DNA sequence encoding a target RNA and a DNA sequence encoding an RNase inhibitor.
  • the RNase inhibitor is selected from E.coli RNase inhibitor RraA and human RNase inhibitor RNH1.
  • the target RNA comprises an affinity tag which is more preferably an aptamer and even more preferably an aptamer is capable of binding to Sephadex.
  • the target RNA further comprises a ribozyme sequence.
  • the DNA sequence encoding the target RNA is under the control of a promoter selected from the group consisting of T3 promoter, T7 promoter, SP6 promoter and tac promoter.
  • the vector further comprises a terminator selected from the T7 terminator, the VSV terminator, the PTH terminator, the rrnB T1 downstream terminator, the rrnC terminator, the concatemer junction sequence of the replicating T7 DNA,the rrnBT1T2 terminator and a variant of any of the foregoing.
  • the present invention is further directed to a host cell comprising the vector comprising a DNA sequence encoding a target RNA and a DNA sequence encoding an RNase inhibitor as further defined above.
  • the host cell comprises a first vector comprising a DNA sequence encoding a target RNA and a second vector comprising a DNA sequence encoding an RNase inhibitor.
  • the RNase inhibitor may be selected from E.coli RNase inhibitor RraA and human RNase inhibitor RNH1.
  • the host cell is a bacterium, more preferably it is E.coli.
  • the host cell preferably E.coli
  • the host cell is capable of expressing a polymerase which may beselected from T3 polymerase, T7 polymerase and SP6 polymerase.
  • a polymerase which may beselected from T3 polymerase, T7 polymerase and SP6 polymerase.
  • the expression of the polymerase is controlled by an operator such as the lac operator.
  • the E.coli may be selected from the group consisting of DH5 ⁇ , BL21, JM109, HMS174, B834, SCS 110, XL1 Blue and XL10 Gold and preferably it isis selected from the group consisting of BL21(DE3), JM109(DE3), HMS174(DE3) and B834(DE3).
  • the present invention is further directed to the use of a host cell as defined above for the in vivo transcription of a DNA sequence encoding a target RNA into said target RNA.
  • the present invention is further directed to the use of a vector comprising a DNA sequence encoding an RNase inhibitor in a process for the in vivo transcription of a DNA sequence encoding a target RNA.
  • the RNase inhibitor may be selected from E.coli RNase inhibitor RraA and human RNase inhibitor RNH1.
  • the process according to the present invention is an in vivo transcription process of a DNA sequence encoding a target RNA.
  • the process for producing a target RNA in a host cell comprises the steps of:
  • the host cell in which the target RNA is produced by in vivo transcription of the DNA sequence encoding the target RNA may be any host cell which can be genetically modified to produce the target RNA and includes both prokaryotic and eukaryotic cells such as fungal cells, plant cells and animal cells.
  • the host cell is a microorganism.
  • a microorganism is a microscopic living organism, which may be single celled or multicellular. Microorganisms are very diverse and include all the bacteria and archaea and almost all the protozoa. They also include some fungi, algae, and certain animals, such as rotifers.
  • the most suitable microorganism or host cell to be used in the process according to the present invention is a bacterial cell or strain.
  • Suitable bacterial strains include for instance Escherichia coli ( E.coli ), Corynebacterium, Lactococcus lactis, Streptomyces, Bacillus subtilis and Pseudomonas fluorescens.
  • E.coli Escherichia coli
  • Corynebacterium for instance Escherichia coli
  • Lactococcus lactis Lactococcus lactis
  • Streptomyces Streptomyces
  • Bacillus subtilis Bacillus subtilis
  • Pseudomonas fluorescens Pseudomonas fluorescens.
  • many additional bacterial strains are known to be suitable for use in recombinant technology.
  • the most preferred bacterial strain to be used in the present invention is an E.coli strain.
  • Preferred E.coli strains to be used in the present invention include strains like DH5 ⁇ , BL21, JM109, HMS174, B834, SCS110, XL1 Blue and XL10 Gold which are commercially available.
  • Preferred microorganisms are those that are able to additionally express a recombinant polymerase, like bacterial strains with recombinant RNA polymerase activity.
  • bacterial strains like E.coli strains, are preferred that are able to express a recombinant polymerase selected from T3 polymerase, T7 polymerase and SP6 polymerase.
  • E.coli strains are the DE3-modified strains of the strains mentioned above, derived from lambda phage DE3 carrying the T7 RNA polymerase gene under the control of the lac operator, such as BL21(DE3), JM109(DE3), HMS174(DE3) and B834(DE3).
  • common yeast expression systems like Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris can also be used by simple adaptation of the process of the present invention to achieve in vivo RNA transcription in the corresponding host cell.
  • microorganisms within the meaning of the present invention include fungal-based systems, particularly filamentous fungi like those commonly used in recombinant expression of proteins, like Aspergillus spp., Trichoderma reesei, and Neurospora crassa.
  • host cells eukaryotic cell lines like human embryonal kidney (HEK) cells or Chinese hamster ovary (CHO) cells, or insect cells like Spodoptera frugiperda Sf9, Sf21 or High Five insect cells that are commonly used in recombinant expression of peptides or proteins.
  • HEK human embryonal kidney
  • CHO Chinese hamster ovary
  • insect cells like Spodoptera frugiperda Sf9, Sf21 or High Five insect cells that are commonly used in recombinant expression of peptides or proteins.
  • plant cells can be used as host cells in the present invention to produce the target RNA by in vivo transcription of the DNA sequence encoding said target RNA.
  • the DNA sequence encoding the target RNA to be produced by the process of the present invention is cloned into an appropriate vector system that allows the transcription of the DNA sequence encoding the target RNA in a host cell, preferably a bacterial system after transfection/transformation.
  • Typical vectors to be used in the present invention are those that are commonly used in standard cloning and transcription procedures in the corresponding host cell.
  • the term "vector” refers to a nucleic acid molecule, preferably to an artificial nucleic acid molecule.
  • a vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame.
  • Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc.
  • a storage vector is a vector, which allows the convenient storage of a nucleic acid molecule, for example, of an mRNA molecule.
  • the vector may comprise a sequence corresponding, e.g., to a desired mRNA sequence or a part thereof, such as a sequence corresponding to the open reading frame and the 3'-UTR of an mRNA.
  • An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.
  • an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a promoter sequence, e.g. an RNA polymerase promoter sequence.
  • a cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector.
  • a cloning vector may be, e.g., a plasmid vector or a bacteriophage vector.
  • a transfer vector may be a vector, which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors.
  • a vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector.
  • a vector is a DNA molecule.
  • a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication.
  • a vector in the context of the present application is a plasmid vector.
  • a recombinant vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual .
  • a variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments, and can be readily determined by persons skilled in the art.
  • the vectors may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells. Coding sequences for selectable markers and reporter genes are well known to the person skilled in the art.
  • the vectors of the present invention may comprise a recognition sequence for one or more restriction endonucleases.
  • Suitable vectors for cloning the target RNA to be produced according to the process of the present invention must include appropriate promoter and terminator regions as well as insertion/cloning sites for the DNA sequence encoding the target RNA, and optionally, for the DNA sequence encoding an RNase inhibitor. Suitable vectors also need to include a selectable marker gene that allows the straight-forward identification of positively transformed clones.
  • Preferred promoter regions in the vector are selected from the group consisting of T3 promoter, T7 promoter, T71ac promoter, SP promoter and tac promoter.
  • Preferred terminator regions in the vector are selected from T7 terminator, the VSV terminator, the PTH terminator, the rrnB T1 downstream terminator, the rrnC terminator, the concatemer junction sequence of the replicating T7 DNA, the rrnBT1T2 terminator and a variant of any of the foregoing.
  • the vector may further contain operator sequences to which a regulator protein binds and influences the affinity of the RNA polymerase to the promoter and therefore transcription.
  • a regulator protein binds and influences the affinity of the RNA polymerase to the promoter and therefore transcription.
  • an operator is the lac operator which is regulated by lactose or a synthetic lactose analogon such as IPTG.
  • Preferred cloning vectors include the plasmids from the pUC or the pET(+) series, particularly the pET21(+) series (Novagen), pET-Duet-1 (Novagen), or pUCminusMCS (Blue Heron). When it is desired that the optional RNase inhibitor is co-expressed in vivo, one preferred vector is pET-Duet-1 (Novagen).
  • plasmids can also be used: pT7Ts (GeneBank Accession No. U26404), vectors from the pGEM®series, for example pGEM®-1 (GeneBank Accession No. X65300) and pSP64 (GeneBank-Accession No. X65327).
  • the in vivo transcription of the DNA sequence encoding the target RNA occurs without subsequently translating the target RNA, e.g. the formed messenger target RNA, into the protein that is optionally encoded by the target RNA.
  • the formation of the protein from the target RNA transcript after in vivo transcription can be preferably prevented by partially or completely deleting or mutating the Shine-Dalgarno sequence in the vector used for transcribing the RNA so that the formed target RNA will not be translated in the bacterial cell in vivo due to the absence of a functional ribosomal binding site that is required to bind the ribosome and initiate translation of the RNA to the encoded protein.
  • the Shine-Dalgarno sequence is AGGAGG, and in E.coli, the sequence is known to be AGGAGGU.
  • the vector used for transcribing the RNA may include a Kozak sequence which is necessary for ribosome binding in eukaryotic cells, in particular if the target RNA is an mRNA which is to be translated in eukaryotic cells.
  • the cells are treated with an antibiotic which inhibits translation of the RNA into protein.
  • Antibiotics inhibiting the translation are known to the skilled person and include chloramphenicol, tetracycline, quinupristin/dalfopristin, fusidic acid, clindamycin and puromycin.
  • the antibiotic which inhibits translation of the RNA into protein is different from the antibiotic used for selecting stably transformed cells.
  • the concentration of the antibiotic is such that it inhibits the cell's anabolic pathways, but does not cause the cells to die.
  • a compound capable of inhibiting the endogenous RNA polymerase of the microorganism but not the RNA polymerase transcribing the target RNA from the vector e.g. the T7 polymerase
  • a compound capable of inhibiting the endogenous RNA polymerase of the microorganism but not the RNA polymerase transcribing the target RNA from the vector e.g. the T7 polymerase
  • One preferred compound is a rifamycin-type compound such as rifampicin, rifabutin and rifapentine.
  • a particularly preferred rifamycin-type compound is rifampicin.
  • an additional DNA sequence encoding at least one RNase inhibitor is cloned into the vector comprising the DNA sequence encoding the target RNA.
  • RNase inhibitor within the meaning of the present invention is a protein which binds to an RNase, thereby inibiting endonucleolytic cleavage of RNA catalyzed by said RNase.
  • the cloning strategy for the DNA sequence encoding the RNase inhibitor must be designed to allow the in vivo expression of a functional RNase inhibitor in the host cell in which the target RNA transcript is formed.
  • the expression of a functional RNase inhibitor in the microorganism in which the target RNA transcript is to be formed is achieved by providing the microorganism with a second vector comprising a DNA sequence encoding an RNase inhibitor.
  • DNA sequence encoding an RNase inhibitor may be integrated into the genome of the host cell.
  • RNase inhibitors are known in the art and in the present invention every possible RNase inhibitor can be used, preferably those that are capable of inhibiting the endogenous RNases of the microorganism used for the in vivo transcription of the target RNA, in particular E.coli RNase inhibitors.
  • Suitable RNase inhibitors are selected from the group consisting of E.coli RNase inhibitors RRaA, RraB and YmdB and human RNase inhibitor RNH1 and combinations thereof.
  • the E.coli RNase inhibitors RRaA and RraB are known to inhibit Ribonuclease E (RNase E) of E.coli ( Lee et al. (2003) Cell 114(5): 623-634 ; Gao et al. (2006) Mol. Microbiol.
  • the E.coli RNase inhibitor YmdB inhibits RNase III ( Kim et al. (2008) Genes Dev. 22: 3497-3508 ) Most preferred is human RNase inhibitor RNH1.
  • the nucleotide sequence of RRaA is available under Uniprot Accession Number POA8R0 and the nucleotide sequence of RNH1 is available under Uniprot Accession Number P13489.
  • the vectors of the present invention are introduced into the microorganism by conventional transformation or transfection techniques.
  • transformation and “transfection” refer to techniques for introducing foreign nucleic acid into the microorganism (or the host cell) and include calcium chloride transformation, lipofection and electroporation. Suitable methods for transforming or transfecting bacterial cells can for example be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989 )), and other laboratory manuals. Methods for introducing DNA into plant cells or mammalian cells in vivo are also well-known to the skilled person.
  • a gene that encodes a selectable marker (such as resistance to antibiotics) may be introduced into the microorganism along with the DNA sequence encoding the target RNA and the DNA sequence encoding the RNase inhibitor.
  • selectable marker is used broadly to refer to markers which confer an identifiable trait to the indicator cell. Non-limiting examples of selectable markers include markers affecting viability, metabolism, proliferation, morphology and the like.
  • Preferred selectable markers include those that confer resistance to drugs, such as ampicillin, kanamycin, tetracycline and chloramphenicol.
  • Nucleic acids encoding a selectable marker may be introduced into a host cell on the same vector as that encoding the target RNA or may be introduced on a separate vector. Cells transfected with the introduced nucleic acid may be identified by drug selection (cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • the host cell transformed with the vector comprising the DNA encoding the target RNA is fermented to produce the target RNA.
  • fermenting means that the host cell is incubated in a medium providing all compounds necessary for cell growth and RNA and protein production at suitable conditions, e.g. a suitable temperature such as 37°C.
  • the transcription of the target RNA may be induced by the addition of an inducing compound, preferably isopropyl- ⁇ -D-1-thiogalactopyranoside (IPTG), if either the DNA encoding the target RNA itself or the DNA encoding the RNA polymerase is linked to a suitable operator such as the lac operator.
  • IPTG isopropyl- ⁇ -D-1-thiogalactopyranoside
  • the bacterial cell is subjected to a fermentation process in order to reach a suitable cell density of the bacterial cells that allows a maximum level of viability and production of the target RNA transcript.
  • the cell density at the time of induction is comparable to cell densities that are typically used in recombinant protein expression using E.coli. Accordingly, the cell culture density OD 600 (measured with a spectrophotometer) should be in the range of 0.4 to 0.8, preferably 0.5 to 0.7, and most preferably at about 0.6. Typically, the growth of the cell culture should be in the log-phase or exponential phase.
  • the preferred substance for inducing the transcription is isopropyl- ⁇ -D-1-thiogalactopyranoside (IPTG) though it is possible to use alternative inducing substances.
  • IPTG isopropyl- ⁇ -D-1-thiogalactopyranoside
  • IPTG IP glycoprotein kinase
  • concentrations of IPTG are typically used in protein expression by E.coli strains.
  • Typical IPTG concentrations are in the range of 0.1 mM to 1.5 mM, preferably from 0.2 to 1.2 mM, and more preferably from 0.5 to 1.0 mM, based on the total cell culture.
  • Preferred concentrations of IPTG include 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM and 1.0 mM
  • the transcription is preferably induced at room temperature, e.g. at a temperature of about 21°C.
  • induction temperatures can be selected below room temperature, for instance below 21°C, or even below 19°C, or even below 17°C.
  • the production period i.e. the period from inducing transcription until obtaining the target RNA
  • the production period is preferably selected in the medium range in order to achieve an appropriate balance between the maximum level of transcription on the one hand and the decreasing stability of the formed target RNA with increasing temperatures on the other hand. Accordingly, a preferred production period until obtaining the target RNA is in the range of 2 to 24 hours, more preferably in the range of 3 to 12 hours, and even more preferably in the range of 4 to 6 hours.
  • the target RNA may be stabilized during the process of in vivo transcription in order to avoid its degradation until it is obtained in isolated form, e.g. by adding special reagents to the crude mixture to preserve and stabilize the target RNA transcripts formed in vivo in the host cell, preferably in the bacterial cell culture. It is also especially important to prevent degradation of the target RNA after production phase when the crude mixture is stored before work-up and before obtaining the target RNA by carrying out subsequent purification procedures.
  • reagents for preventing gene expression and stabilization of formed transcripts of the target RNA are commercially available, such as RNAprotect cell reagent or RNA protect Bacteria Reagent, both available from Qiagen.
  • the addition of the above compounds during fermentation for stabilizing the target RNA and maximizing the amount of target RNA formed in the in vivo transcription process of the target RNA can be started and ended at certain phases of fermentation in order to optimize the stabilizing effect of such treatment.
  • the skilled person will be able to establish the best scheme of addition for maximum efficiency of such treatment based on routine testing.
  • the target RNA is obtained.
  • the target RNA is specifically obtained, meaning that after the process of the present invention the target RNA is essentially free from the endogenous RNA of the host cell used for producing the target RNA.
  • Essentially free means that less than 5% or 4%, preferably less than 3% or 2%, more preferably less than 1.5% or 1% and most preferably less than 0.8% or 0.5% endogenous RNA of the microorganism used for producing the target RNA is present in the target RNA obtained by the process of the present invention.
  • the target RNA is obtained from the cytoplasm of the microorganism, since it is not secreted into the cell culture medium.
  • the basic steps of obtaining the target RNA include first separating of the host cells in which the target RNA has been formed by in vivo transcription from the cell culture medium, then washing the host cells to remove cell culture medium, followed by lysis of the host cells and further purifying the target RNA by separating it from cell debris, endogenous proteins of the host cell and endogenous DNA and RNA of the host cell until the isolated target RNA is obtained.
  • the host cells preferably bacterial cells
  • the host cells are harvested by separating them from the cell culture medium.
  • One preferred method for the separation of the host cells, preferably the bacterial cells, from the culture medium is centrifugation by which the cells are obtained as cell pellet which can be processed further.
  • Cell lysis is preferably carried out by enzymatic lysis, mechanical lysis and/or chemical lysis. There are many efficient techniques available in the art to efficiently disrupt the host cells. These techniques are known to the skilled person. For instance, enzymatic lysis may be carried out by using lysozyme. Mechanical or physical lysis includes mechanical disruption, liquid homogenization, the application of high frequency sound waves, e.g. ultrasonification, the application of freeze/thaw cycles and manual grinding.
  • the RNA is further purified. Typically, in a first step the total RNA of the host cell is separated from other molecules within the host cell and then in a second step the target RNA is specifically obtained.
  • RNA isolation methods can be preferably applied like for instance the phenol/chloroform extraction and precipitation of RNA with which the skilled person will be familiar as one standard procedure in the field of recombinant RNA technology.
  • kit products which are commercially available for the isolation of RNA can be used.
  • kit products are based on special solid matrices that are capable of binding RNA molecules with relatively high selectivity.
  • kit is the RNeasy Mini Kit® (Qiagen).
  • RNA purification methods useful for isolation of RNA are preferably selected from cation exchange chromatography, anion exchange chromatography, membrane absorbers, reversed phase chromatography, normal phase chromatography, size exclusion chromatography, hydrophobic interaction chromatography, mixed mode chromatography, affinity chromatography, hydroxylapatite (HA) chromatography, or combinations thereof.
  • the target RNA is further obtained from the microorganism by separating the target RNA from the endogenous RNA of the microorganism.
  • separating the target RNA from the endogenous RNA of the microorganism comprises depleting the ribosomal RNA from the crude mixture.
  • the ribosomal RNA is preferably depleted by capture hybridization of the ribosomal RNA with oligonucleotides that have the ability to selectively bind ribosomal RNA, particularly bacterial 16 S and 23S ribosomal RNA and that are immobilized on a solid phase.
  • One possibility is to deplete the ribosomal RNA in the crude mixture by the use of commercially available kits like the MICROBExpressTM kit (Ambion) which is based on a capture hybridization approach using magnetic beads.
  • the target RNA can be obtained from the crude mixture by affinity chromatography in which the target RNA is trapped by a solid or stationary phase or medium which has selective affinity to the target RNA.
  • a solid or stationary phase or medium which has selective affinity to the target RNA.
  • One preferred stationary phase is a Sephadex column carrying an immobilized ligand with affinity to the target RNA.
  • Other molecules in the crude mixture will not be bound to the ligand, since these molecules lack the required affinity.
  • the stationary phase with the target RNA bound to the ligand can then be removed from the crude mixture, washed and the target RNA is obtained again in enriched form after removing the target RNA from the affinity ligand.
  • the target RNA can be removed by a change of salt concentration, ionic strength, pH value and the like.
  • elution can be accomplished by application of a pH or salt gradient to the stationary phase.
  • the use of affinity chromatography to obtain the target RNA after in vivo transcription in the microorganism is preferred because the target RNA can be obtained in a straight-forward manner avoiding multiple consecutive purification steps that normally lead to considerable loss of product due to continued slow degradation and accumulation of product losses along a series of consecutive purification steps.
  • an affinity purification scheme is based on immobilizing a nucleic acid ligand on the stationary phase or solid matrix which is capable of hybridizing with the target RNA via the conventional rules of base-pairing.
  • the DNA ligand will have a nucleic acid sequence which is sufficiently complementary to bind the target RNA with a strength which is sufficient to maintain binding during the washing steps.
  • the target RNA comprises an affinity tag which is capable of binding to an affinity matrix and which can be removed from the target RNA after purification.
  • the affinity tag may be attached to the target RNA via a linker between the target RNA and the affinity tag sequence.
  • the linker may comprise 10 nucleotides or less and may comprise any nucleotides or combinations thereof.
  • the affinity tag comprises an additional RNA sequence which is capable of binding to a matrix e.g. Sephadex or to a protein and/or peptide with high selectivity.
  • the affinity tag preferably comprises the additional RNA sequence upstream or downstream of the target RNA sequence.
  • the additional RNA sequence is an aptamer.
  • Aptamers are short oligonucleotides usually comprising 25 to 70 nucleotides which are capable of binding to a specific molecule due to their specific 3D structure.
  • suitable aptamers for use in the present invention are those that bind to a structure useful for purification such as Sephadex aptamer, glutathione aptamer, cellulose aptamer, Cibacron blue aptamer and streptavidin aptamer ( Srisawat and Engelke (2001) RNA 7: 632-641 ) which are able to bind to common chromatography matrices.
  • the aptamer is capable of binding to Sephadex.
  • Such an aptamer is described in Srisawat et al. (2001) Nucl. Acids Res. 29(2) e4 and has a core sequence of GAGUAAUUUACG (SEQ ID No. 9).
  • the target RNA further comprises an activatable ribozyme sequence, wherein the activatable ribozyme sequence is preferably inserted between the DNA sequence encoding the target RNA and the DNA sequence encoding the additional RNA sequence in order to allow subsequent release of the target RNA from the additional RNA sequence used as affinity tag by triggering autocleavage of the activatable ribozyme sequence.
  • a ribozyme is a catalytic nucleic acid molecule which is an RNA molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage of other nucleic acid molecules such as RNA molecules.
  • the term ribozyme is used interchangeably with terms such as catalytic RNA, enzymatic RNA, or RNA enzyme.
  • RNA molecules which are capable of catalyzing reactions in the absence of any protein component and these molecules were called ribozymes.
  • ribozymes Several classes of ribozymes occurring in natural systems have been discovered, most of which catalyse intramolecular splicing or cleavage reactions (reactions ' in cis '). Since most of the naturally occurring ribozymes catalyse self-splicing or self-cleavage reactions, it was necessary to convert them into RNA enzymes which can cleave or modify target RNAs without becoming altered themselves (reactions ' in trans ') .
  • Ribozymes are broadly grouped into two classes based on their size and reaction mechanisms: large and small ribozymes.
  • the first group consists of the self-splicing group I and group II introns as well as the RNA component of RNase P, whereas the latter group includes the hammerhead, hairpin, hepatitis delta ribozymes and varkud satellite (VS) RNA as well as artificially selected nucleic acids.
  • Large ribozymes consist of several hundreds up to 3000 nucleotides and they generate reaction products with a free 3'-hydroxyl and 5'-phosphate group.
  • Group I introns include the self-splicing intron in the pre-ribosomal RNA of the ciliate Tetrahymena thermophilia. Further examples of group I introns interrupt genes for rRNAs, tRNAs and mRNAs in a wide range of organelles and organisms. Group I introns perform a splicing reaction by a two-step transesterification mechanism: The reaction is initiated by a nucleophilic attack of the 3'-hydroxyl group of an exogenous guanosine cofactor on the 5'-splice site.
  • the free 3'-hydroxyl of the upstream exon performs a second nucleophilic attack on the 3'-splice site to ligate both exons and release the intron.
  • Substrate specificity of group I introns is achieved by an Internal Guide Sequence (IGS).
  • IGS Internal Guide Sequence
  • the catalytically active site for the transesterification reaction resides in the intron, which can be re-engineered to catalyse reactions in trans.
  • Group II introns are found in bacteria and in organellar genes of eukaryotic cells. They catalyse a self-splicing reaction that is mechanistically distinct from group I introns because they do not require a guanosine cofactor. Instead, the 2'-hydroxyl of a specific adenosine at the so-called branch site of the intron initiates the reaction by a nucleophilic attack on the splice-site to form a lariat-type structure.
  • RNase P was the first example of a catalytic RNA that acts in trans on multiple substrates.
  • RNase P can be considered to be the only true naturally occurring trans-cleaving RNA enzyme known to date. However, for full enzymatic activity under in vivo conditions the protein component is essential.
  • the hammerhead ribozyme is found in several plant virus satellite RNAs, viroids and transcripts of a nuclear satellite DNA of newt. This ribozyme is the smallest of the naturally occurring ribozymes and processes the linear concatamers that are generated during the rolling circle replication of circular RNA plant pathogens.
  • the development of hammerhead variants that cleave target RNA molecules in trans was a major advancement that made possible the use of ribozyme technology for practical applications.
  • the hammerhead ribozyme motif that has widely been applied since then comprises three helical sections connected via a three-way helical junction.
  • catalytic entity In hairpin ribozymes the catalytic entity is part of a four-helix junction.
  • a minimal catalytic motif containing approximately 50 nucleotides has been identified that can be used for metal-ion dependent cleavage reactions in trans. It consists of two domains, each harbouring two helical regions separated by an internal loop, connected by a hinge region. One of these domains results from the association of 14 nucleotides of a substrate RNA with the ribozyme via base-pairing.
  • the hepatitis delta virus (HDV) ribozyme is found in a satellite virus of hepatitis B virus. Both the genomic and the antigenomic strand express cis-cleaving ribozymes of ⁇ 85 nucleotides that differ in sequence but fold into similar secondary structures. The crystal structure of the ribozyme reveals five helical regions are organized by two pseudoknot structures. The catalytic mechanism of the hepatitis delta virus ribozyme appears to involve the action of a cytosine base within the catalytic centre as a general acid-base catalyst. The hepatitis delta ribozyme displays high resistance to denaturing agents like urea or formamide. Trans-cleaving derivatives of this ribozyme have been developed.
  • the Varkud Satellite (VS) ribozyme is a 154 nucleotide long and is transcribed from a plasmid discovered in the mitochondria of certain strains of Neurospora.
  • the VS ribozyme is the largest of the known nucleolytic ribozymes
  • An activatable ribozyme may be activated by a variety of ways, including by effector molecules and/or other means such as radiation (e.g., light radiation, UV radiation, IR radiation), as well as temperature and/or pH changes.
  • the activatable ribozyme is activated by the addition of Mg2+.
  • Activatable ribozymes are well known in the art and include, for example, those described in Koizumi et al., (1999) Nature Struct. Biol. 6(11): 1062-1071 which are activatable by specific nucleoside 3',5'-cyclic monophosphate compounds such as cGMP or cAMP, those described in Grate and Wilson, (1999) Proc. Nat. Acad. Sci 96: 6131-6136 (a malachite green (MG)-tagged RNA that cleaves upon laser irradiation) as well as the Glucosamine-6-phosphate activated (GlmS) ribozyme which is activated by glucosamine-6-phosphate (GlcN6P).
  • MG malachite green
  • GlmS Glucosamine-6-phosphate activated
  • activatable ribozymes include virus-derived activatable ribozymes such as activatable hepatitis delta virus ribozyme (H5V) ( Shih et al., Annual Review of Biochemistry 71: 887-917 (2002 )).
  • the above-mentioned activatable ribozyme sequence is a sequence of a glmS ribozyme, which is typically produced by Gram-positive bacteria such as Bacillus subtilis and Bacillus anthracis ( Roth, A. et al. (2006) RNA 12: 607-619 ).
  • the above-mentioned glmS ribozyme is a Bacillus anthracis glmS ribozyme sequence.
  • activatable ribozymes may be generated by combining a ribozyme and a riboswitch, as described in Chen and Elington, PLoS Comput Biol 2009 5(12): e1000620 .
  • the additional RNA nucleotide sequence of the affinity tag is an bacteriophage boxB RNA sequence which is an RNA hairpin sequence comprising short stems of typically 5 to 7 base pairs with a 5 or 6 nucleotide loop as found in some bacteriophages, preferably those of the Lambda family of bacteriophages like lambda, ⁇ 21 and P22.
  • bacteriophage boxB RNA sequences have a strong affinity for specific phage-derived polypeptides/peptides involved in the regulation of transcription (e.g., transcription termination, transcription anti-termination), such as bacteriophage N proteins and Coliphage HK022 nun protein, and more particularly for the amino-terminal region of such polypeptides/peptides.
  • bacteriophage boxB RNA sequences are described in Cilley and Williamson RNA (2003) 9: 663- 676 ; Neely and Friedman, Molecular Microbiology (2000) 38(5): 1074-1085 and Chattopadhyay et al. (1995) Proc. Natl. Acad. Sci. 92: 4061 -4065 .
  • the above-mentioned bacteriophage boxB RNA sequence is a bacteriophage lambda ( ⁇ ) boxB RNA sequence.
  • the above-mentioned bacteriophage boxB RNA sequence and/or activatable ribozyme sequence may be incorporated at the 5' or 3' end of the target RNA sequence, or within the target RNA sequence.
  • the above-mentioned bacteriophage boxB sequence and/or activatable ribozyme sequence is/are incorporated at the 3' end of the target RNA sequence, preferably in a way that the activatable ribozyme sequence is inserted between the target RNA and the bacteriophage boxB sequence.
  • the above-mentioned method further comprises incorporating a linker at the 3' end of said target RNA (e.g. between the 3' end of the target RNA and the affinity tag sequence).
  • the linker comprises 10 nucleotides or less and comprises any nucleotides or combinations thereof.
  • the linker comprises 5 nucleotides or less, and in a further embodiment it comprises 1 or 2 nucleotides.
  • the above-mentioned bacteriophage boxB sequence may be incorporated at the 5' end, at the 3' end or within the activatable ribozyme sequence. In a preferred embodiment, the above-mentioned bacteriophage boxB sequence is incorporated at the 3' end of the activatable ribozyme sequence. In another embodiment, the above-mentioned bacteriophage boxB sequence is incorporated into a stem-loop of the activatable ribozyme sequence, in a further embodiment into the variable apical P1 stem-loop of the activatable ribozyme sequence.
  • the affinity tag on the target RNA sequence comprising the activatable ribozyme sequence and a bacteriophage boxB RNA sequence as decribed above may be immobilized on the solid matrix by binding to a peptide/polypeptide construct comprising a) a boxB RNA binding peptide, b) an immobilizing moiety and, optionally, c) a peptide linker linking the boxB RNA binding peptide and the immobilizing moiety.
  • boxB RNA binding peptide refers to phage-derived peptides capable of binding with high affinity to a bacteriophage boxB RNA sequence as defined above.
  • these phage-derived peptides are derived from specific regions of proteins (generally arginine-rich motifs located in the N-terminal portion) involved in the regulation of transcription (e.g. termination), such as bacteriophage N proteins and coliphage HK022 Nun proteins.
  • bacteriophage N peptide refers to an arginine-rich peptide motif (ARMs) derived from bacteriophage N proteins and having affinity for a bacteriophage boxB RNA.
  • ARMs arginine-rich peptide motif
  • Examples of bacteriophage N peptides are disclosed in Cilley and Williamson, RNA (2003) 9: 663-676 ; Su et al., Biochemistry (1997), 36: 12722-12732 and Austin et al., 2002, supra.
  • Bacteriophage N peptide as used herein also encompass derivatives of naturally occurring or mutated N peptides, for example acridine derivatives as described in Qi et al., Biochemistry (2010) 49: 5782-5789 .
  • the above-mentioned boxB RNA binding peptide is a peptide derived from Coliphage HK022 Nun protein, and more particularly comprises residues 1 to 44, e.g. residues 10-44 or 20-44, of the Coliphage HK022 Nun protein ( Faber et al. J. Biol. Chem. 276(34): 32064-32070 ).
  • the above-mentioned boxB RNA binding peptide binds to said bacteriophage boxB with a dissociation constant (K D ) of about 2 x 10 -8 M or less at physiological salt concentrations (about 150 mM).
  • the above-mentioned boxB RNA binding peptide binds to said bacteriophage boxB with a K D of about 5 x 10 -8 M or less, about 2 x 10 -8 M or less, 1 x 10 -8 M or less, about 5 x 10 -9 M or less, about 1 x 10 -9 M or less, or about 1 x 10 -10 or less at physiological salt concentrations (about 150 mM).
  • the above-mentioned boxB RNA binding peptide comprises from about 10 to about 40 amino acids, in further embodiments from about 10 to about 30, from about 13 to about 25, from about 15 to about 21 amino acids, from about 17 to about 20 amino acids.
  • the above-mentioned peptide/polypeptide construct comprises a plurality of boxB RNA binding peptides (e.g. bacteriophage N peptide), either as multiple copies of the same peptide, or as different peptides.
  • the plurality of boxB RNA binding peptides may be covalently linked either directly (e.g. through a peptide bond) or via a suitable linker moiety, e.g. a linker of one or more amino acids (e.g. a polyglycine linker or the like) or another type of chemical linker (e.g.
  • the above-mentioned boxB RNA binding peptide (e.g. bacteriophage N peptide) comprises at its carboxy-terminal end a peptide linker that links the bacteriophage N peptide and the moiety capable of binding to the solid support (immobilizing or binding moiety).
  • the configuration of the peptide/polypeptide construct (preferably from N- to C-terminus) binding the target RNA with the activatable ribozyme sequence and the additional RNA nucleotide sequence (being preferably the bacteriophage boxB RNA sequence) as decribed above is as follows: boxB RNA binding peptide - peptide linker - immobilizing moiety
  • boxB binding peptide has already been described above.
  • the peptide linker may be any amino acid sequence, such as a natural (e.g. a naturally occurring peptide or polypeptide) or an artificial (e.g. a synthetic, non-naturally occurring peptide or polypeptide) sequence, that does not significantly interfere with the binding of the boxB RNA binding peptide to the bacteriophage boxB RNA sequence) and that permits the binding of the immobilizing moiety to the solid support (e.g. that does not significantly interfere with the binding of the immobilizing moiety to the solid support).
  • the above-mentioned construct further comprises a moiety capable of binding to said solid support (an immobilizing or binding moiety).
  • the immobilizing moiety is linked to the peptide linker and is thus indirectly fused (i.e. through the peptide linker) to the C- terminus of the boxB RNA binding peptide (e.g. bacteriophage N peptide).
  • the immobilizing moiety may be any moiety capable of binding, either directly or indirectly, to a solid support.
  • the solid support comprises a moiety or ligand capable of binding to the immobilizing moiety of the polypeptide construct.
  • a polyhistidine tag (commonly referred to as His-tag) is a moiety comprising a plurality of histidine residues (at least 5, typically 6) which are capable of binding a solid support that contains bound metal ions, and more particularly nickel or cobalt.
  • binding moieties useful for affinity purification include biotin-based tags (which binds to avidin, streptavidin or analogs/derivatives thereof), Strep tag (a short peptide of 8 amino acids) which binds to Sfrep-TactinTM an engineered form of streptavidin (commercially available from Qiagen), as well as Glutathione S-transferase (GST) tag, which binds to a glutathione-containing solid support.
  • Any affinity tag-based system may be used in the constructs and methods of the present invention.
  • the target RNA is preferably obtained by specifically binding the target RNA to a solid support via the affinity tag which may either comprise an aptamer or a bacteriophage boxB RNA sequence.
  • solid support generally refers to any material to which a biospecific ligand is covalently attached, such as chromatographic media (e.g. resin, gel, beads) that are generally used to purify molecules and macromolecules based on various properties (e.g. size, charge, affinity for a given ligand, etc.).
  • Solid supports are well known in the art and include, for example, matrices of polyacrylamide resins or cross-linked polysaccharides such as cross-linked dextran (e.g. SephadexTM), cross-linked agarose (e.g. SepharoseTM) and the like.
  • binding to the solid support may for example be achieved by batch treatment or column chromatography.
  • Batch treatment typically entails combining the sample (containing the target RNA of the present invention) with the solid support in a vessel to allow binding of the target RNA, mixing, separating the solid support (e.g. by centrifugation), removing the liquid phase, washing, separating the solid support (e.g., re- centrifuging), adding an elution buffer, separating the solid support (e.g. re-centrifuging) and removing the eluate.
  • separating the solid support e.g. by centrifugation
  • removing the liquid phase washing
  • washing separating the solid support (e.g., re- centrifuging)
  • adding an elution buffer separating the solid support (e.g. re-centrifuging) and removing the eluate.
  • Column chromatography typically entails packing the solid support onto a chromatography column, passing the sample (containing the target RNA of the present invention) through the column to allow binding of the RNA, passing a wash buffer through the column and subsequently an elution buffer to collect the bound material.
  • Hybrid approaches may also be used, for example binding via a batch method followed by packing the solid support with the bound target RNA onto a column, followed by washing and elution on the column.
  • the batch method can also be combined with the use of spin cups and SteriflipTM filter units, which typically improve resin recovery.
  • the target RNA is obtained in a purity of at least 95% or 96%, preferably of at least 97% or 98%, more preferably of at least 98.5% or 99% and most preferably of at least 99.2% or 99.5%.
  • the purity is determined by calculating the percentage of the target RNA relative to the total RNA obtained by the process of the present invention.
  • the DNA sequence encoding the target RNA sequence to be produced by the present process of in vivo transcription refers to the sequence of nucleotides consisting of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate and deoxycytidine monophosphate monomers which are - by themselves - composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety. These nucleotides polymerise to form a polynucleotide with a characteristic backbone structure.
  • the backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e.
  • deoxyribose of a first and a phosphate moiety of a second, adjacent monomer.
  • the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by AT-base-pairing and G/C-base-pairing.
  • the specific order of the monomers in the DNA sequence i.e. the order of the bases linked to the sugar/phosphate-backbone is called the DNA sequence.
  • the DNA sequence encoding the target RNA refers to the sequence of DNA nucleotides which is complementary to the sequence of the target RNA obtained by transcription of this DNA sequence.
  • RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually adenosine monophosphate, uridine monophosphate, guanosine monophosphate and cytidine monophosphate monomers, which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific succession of the monomers is called the RNA sequence or sequence of RNA. Usually RNA is obtained by transcription of a DNA sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria.
  • the sequence forming the target RNA according to the present invention refers to a RNA polynucleotide sequence which can comprise modified or non-modified (un-modified) nucleotides thereby forming modified or non-modified target RNA depending on the presence or absence of modified RNA nucleotides.
  • the target RNA forms a modified target RNA, otherwise it is a non-modified or un-modified target RNA.
  • the production of non-modified target RNA is preferred in the process of the present invention.
  • non-modified RNA or “non-modified target RNA” refers to a RNA sequence comprising nucleotides which are present in nature.
  • a non-modified reference mRNA corresponds to an mRNA sequence comprising naturally occurring nucleotides, including nucleotides comprising naturally occurring modifications, and/or naturally occurring untranslated regions, such as the naturally occurring 5'-CAP structure m7GpppN, optionally the naturally occurring 5'-UTR, if present in the naturally occurring mRNA sequence, the wild type open reading frame, optionally the naturally occurring 3'-UTR if present in the naturally occurring mRNA sequence and a poly A sequence with approximately 30 adenosines.
  • modified RNA comprises any type of ribonucleic acid molecule that is modified such that the amount of protein or peptide produced from said modified RNA measured at a specific time point or over a specific time period is increased in comparison with RNA lacking the modification ("unmodified reference RNA").
  • unmodified reference RNA Methods for measuring increased protein expression of a modified RNA relative to non-modified RNA are for instance disclosed in WO 2015/024667 A1 which is herein incorporated by reference.
  • RNA modification may refer to chemical modifications comprising backbone modifications as well as sugar modifications, base modifications or lipd modifications.
  • the modified RNA molecule as defined herein may contain nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications.
  • a backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides contained in a nucleic acid molecule as defined herein are chemically modified.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the nucleic acid molecule as defined herein.
  • a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the nucleic acid molecule of the nucleic acid molecule.
  • nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.
  • modified nucleosides and nucleotides which may be incorporated into the modified RNA as described herein, can be modified in the sugar moiety.
  • the 2' hydroxy! group (OH) can be modified or replaced with a number of different "oxy" or “deoxy” substituents.
  • Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g.
  • R H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), -O(CH 2 CH 2 O)nCH 2 CH 2 OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g. by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, wherein the amino group, e.g.
  • NRR can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy.
  • “Deoxy” modifications include hydrogen, amino (e.g. NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified RNA can include nucleotides containing, for instance, arabinose as the sugar.
  • the phosphate backbone may further be modified in the modified nucleosides and nucleotides, which may be incorporated into the modified RNA, as described herein.
  • the phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).
  • modified nucleosides and nucleotides which may be incorporated into the modified RNA, as described herein, can further be modified in the nucleobase moiety.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil.
  • the nucleosides and nucleotides described herein can be chemically modified on the major groove face.
  • the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
  • the nucleotide analogues/modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'-triphosphate, 2-Aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-Amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-Fluorothymidine-5'-triphosphate, 2'-O-Methyl inosine-5'-triphosphate 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine- 5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-Bromo-2'-deoxycy
  • nucleotides for base modifications selected from the group of base- modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'- triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.
  • modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl- pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio- uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza
  • modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2- thio-zebularine, 2-thio-zebul
  • modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethylmethyladeno
  • modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine.
  • the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
  • a modified nucleoside is 5'-O-(1-Thiophosphate)-Adenosine, 5'-O-(1-Thiophosphate)-Cytidine, 5'-O-(1-Thiophosphate)-Guanosine, 5'-O-(1-Thiophosphate)-Uridine or 5'-O-(1-Thiophosphate)-Pseudouridine.
  • the modified RNA may comprise nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2 -amino-purine, Pseudo-iso
  • the target RNA to be produced in the process according to the present invention is broadly defined as follows and can include the following types of RNA.
  • Preferred target RNAs are defined below and include mRNA, viral RNA, retroviral RNA and replicon RNA, bicistronic or multicistronic RNA, small interfering RNA (siRNA), antisense RNA, CRISPR RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).
  • Very preferred target RNA is immunostimulating RNA and/or mRNA. Most preferred target RNA is mRNA.
  • RNA In vivo, transcription of DNA usually results in the so-called premature RNA, which has to be processed into so-called messenger RNA, usually abbreviated as mRNA.
  • Processing of the premature RNA e.g. in eukaryotic organisms, comprises a variety of different post-transcriptional modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA.
  • the mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein.
  • an mRNA found in eukaryotic cells comprises a 5'-cap, optionally a 5'UTR, an open reading frame, optionally a 3'UTR and a poly(A) sequence.
  • the RNA is obtained from bacterial cells, the mRNA has to be enzymatically capped by a capping enzyme. Suitable capping enzymes are known in the art. Aside from messenger RNA, several non-coding types of RNA exist which may be involved in regulation of transcription and/or translation.
  • a “5 '-cap” is an entity, typically a modified nucleotide entity, which generally “caps" the 5'-end of a mature mRNA.
  • a 5'-cap may typically be formed by a modified nucleotide (cap analog), particularly by a derivative of a guanine nucleotide.
  • the 5'-cap is linked to the 5'-terminus of a nucleic acid molecule, preferably an RNA, via a 5'-5'-triphosphate linkage.
  • a 5'-cap may be methylated, e.g. m7GpppN (e.g.
  • a 5'-cap may be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide.
  • the 5'-cap is linked to the 5'-terminus via a 5'-5'-triphosphate linkage.
  • a 5'-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, typically the 5'-end of an RNA.
  • m7GpppN is the 5'-cap structure which naturally occurs in mRNA, typically referred to as capO structure.
  • Enzymes such as cap-specific nucleoside 2'-O-methyltransferase enzyme create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl.
  • Such a structure is called the cap1 structure.
  • 5'cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide, 1 -(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted nucleo
  • CAP1 (methylation of the ribose of the adjacent nucleotide of m7GpppN)
  • CAP2 (methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN)
  • CAP3 (methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN)
  • CAP4 (methylation of the ribose of the 4th nucleotide downstream of the m7GpppN)
  • ARCA anti-reverse CAP analogue, modified ARCA (e.g.
  • phosphothioate modified ARCA inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • a “5'-UTR” is typically understood to be a particular section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Typically, the 5'-UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame.
  • the 5'-UTR may comprise elements for controlling gene expression, which are also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites.
  • the 5'-UTR may be posttranscriptionally modified, for example by addition of a 5'-cap. In the context of the present invention, a 5'-UTR corresponds to the sequence of a mature mRNA, which is located between the 5'-cap and the start codon.
  • the 5'-UTR corresponds to the sequence, which extends from a nucleotide located 3' to the 5'-cap, preferably from the nucleotide located immediately 3' to the 5'-cap, to a nucleotide located 5' to the start codon of the protein coding region, preferably to the nucleotide located immediately 5' to the start codon of the protein coding region.
  • the nucleotide located immediately 3' to the 5'-CAP of a mature mRNA typically corresponds to the transcriptional start site.
  • the term “corresponds to” means that the 5'-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5'-UTR sequence, or a DNA sequence, which corresponds to such RNA sequence.
  • a 5'-UTR of a gene is the sequence, which corresponds to the 5'-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term “5'-UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 5'-UTR.
  • 3'-UTR refers to a part of the nucleic acid molecule, which is located 3' (i.e. "downstream") of an open reading frame and which is not translated into protein.
  • a 3'-UTR is the part of an mRNA, which is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(N/A) sequence of the (m)RNA.
  • ORF open reading frame
  • CDS coding sequence
  • a 3'-UTR of the nucleic acid molecule may comprise more than one 3'-UTR elements, which may be of different origin, such as sequence elements derived from the 3'-UTR of several (unrelated) naturally occurring genes.
  • the term 3'-UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g. a poly(A) sequence.
  • a 3'-UTR of the mRNA is not translated into an amino acid sequence.
  • the 3'-UTR sequence is generally encoded by the gene, which is transcribed into the respective mRNA during the gene expression process.
  • the genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns.
  • the pre-mature mRNA is then further processed into mature mRNA in a maturation process.
  • This maturation process comprises the steps of 5'capping, splicing the pre-mature mRNA to excize optional introns and modifications of the 3'-end, such as polynucleotidylation/polyadenylation of the 3'-end of the pre-mature mRNA and optional endo-/ or exonuclease cleavages etc.
  • a 3'-UTR corresponds to the sequence of a mature mRNA which is located between the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and the poly(A) sequence of the mRNA.
  • the 3'-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3'-UTR sequence, or a DNA sequence, which corresponds to such RNA sequence.
  • a 3'-UTR of a gene is the sequence, which corresponds to the 3'-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term "3'-UTR of a gene” encompasses the DNA sequence and the RNA sequence (both sense and antisense strand and both mature and immature) of the 3'-UTR.
  • the term "3'-UTR element” typically refers to a fragment of a 3'-UTR as defined herein.
  • the term comprises any nucleic acid sequence element, which is located 3' to the ORF in the artificial nucleic acid molecule, preferably the mRNA, according to the invention.
  • the term covers, for example, sequence elements derived from the 3'-UTR of a heterologous gene as well as elements such as a poly(C) sequence or a histone stem-loop.
  • Polyadenylation is typically understood to be the addition of a poly(A) sequence, i.e. a sequence of adenosine monophosphate, to a nucleic acid molecule, such as an RNA molecule.
  • a poly(A) sequence i.e. a sequence of adenosine monophosphate
  • RNA molecule such as an RNA molecule.
  • the term may relate to polyadenylation of RNA as a cellular process as well as to polyadenylation carried out by enzymatic reaction in vitro or by chemical synthesis.
  • a poly(A) sequence also called poly(A) tail or 3'-poly(A) tail, is usually understood to be a sequence of adenine nucleotides, e.g., of up to about 400 adenosine nucleotides, e.g. from about 20 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides, which is preferably added to the 3'-terminus of an mRNA.
  • a poly(A) sequence is typically located at the 3'-end of an RNA, in particular mRNA.
  • a poly(A) sequence may be located within an (m)RNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector.
  • the term 'poly(A) sequence' further comprises also sequence elements, preferably artificial sequence elements, that are part of the 3'-UTR or located at the 3'-terminus of the artificial nucleic acid molecule, and which preferably comprise up to 1100 adenine nucleotides, more preferably at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 500, 600, 700, 800, 900, or at least 1000 adenine nucleotides.
  • the poly(A) sequence consists of adenosine monophosphates.
  • RNA as used herein also encompasses other RNA molecules, such as viral RNA, i.e. RNA derived from a virus, retroviral RNA, i.e. RNA derived from a retrovirus, and replicon RNA.
  • viral RNA i.e. RNA derived from a virus
  • retroviral RNA i.e. RNA derived from a retrovirus
  • replicon RNA RNA molecules
  • RNA typically RNA, preferably an mRNA, that typically has two (bicistronic) or more (multicistronic) open reading frames (ORF).
  • An open reading frame in this context is a sequence of codons that is translatable into a peptide or protein.
  • siRNA small interfering RNA
  • siRNA molecules are 12-28 nucleotides long, more preferably 15-25 nucleotides long, still more preferably 19-23 nucleotides long and most preferably 21-23 nucleotides long. Therefore, preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotides in length.
  • antisense RNA includes any RNA molecule which is suitable as an antisense RNA to regions of RNA, preferably mRNA, and most preferably to regulatory elements thereof, and which is capable of causing inhibition of gene expression by complementary binding to these regions.
  • the antisense RNA may also comprise DNA sequences.
  • CRISPR RNA is derived from CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria as an adaptive immune system to defend against viral attack.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPRs refers to loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea.
  • the CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus.
  • CRISPR RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complementary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (CRISPR RNA).
  • CRISPR RNA Clustered Regularly Interspaced Short Palindromic Repeats
  • Ribozymes are typically RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA target molecules in a nucleotide base sequence specific manner, blocking their expression and affecting normal functions of other genes. Ribozymes can be targeted to virtually any RNA transcript, and achieve efficient cleavage in vitro.
  • RNA aptamer is understood herein to refer to nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
  • RNA aptamer as used herein is an aptamer comprising ribonucleoside units. Aptamers, preferably RNA aptamers, are capable of specifically binding to selected targets and modulating the target's activity. Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties.
  • Riboswitches are RNA elements that reside in the 5' untranslated region (UTR) of genes and modulate their expression using either transcriptional or translational mechanisms ( Roth and Breaker (2009) Annu Rev Biochem 78: 305-334 ).
  • RNA immunostimulatory RNA
  • isRNA may typically be an RNA that is able to induce an innate immune response. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an immune response, e.g. by binding to a specific kind of Toll-like receptor (TLR) or other suitable receptors.
  • TLR Toll-like receptor
  • mRNAs having an open reading frame and coding for a peptide/protein may induce an innate immune response and, thus, may be immunostimulatory RNAs.
  • small nuclear RNA refers to small RNA molecules which are synthesized and/or function in the nucleoplasm and/or the nucleolus of the cell.
  • snoRNAs small nucleolar RNAs
  • box C/D box C/D
  • box H/ACA snoRNAs box C/D
  • box H/ACA snoRNAs box C/D
  • snoRNAs guide sequences that are known to canonically pair with complementary regions on a target pre-rRNA,, forming a RNA duplex and facilitating the enzymatic activity of methylases and uridylases that site-specifically modify pre-rRNA bases by either 2'-0-methylation or pseudouridylation, respectively.
  • Micro RNAs are single-stranded RNAs of typically 22-nucleotides that are processed from about 70 nucleotide hairpin RNA precursors by the RNase III nuclease. Similar to siRNAs, miRNAs can silence gene activity through destruction of homologous mRNA in plants or blocking its translation in plants and animals. These 20-22 nucleotide non-coding RNAs have the ability to hybridize via base-pairing with specific target mRNAs and downregulate the expression of these transcripts, by mediating either RNA cleavage or translational repression. Recent studies have indicated that miRNAs have important functions during development. Further, miRNAs are transcribed by RNA polymerase 11 as polyaclenylated and capped messages known as pri-miRNAs.
  • PiRNA Pore-interacting RNA
  • piRNA is a class of small non-coding RNA molecules that is expressed in, or can be introduced into animal cells (see, e.g., Seto et al. (2007) Molecular Cell, 26(5): 603-609 ; Siomi et al. (2011) Nat. Rev. Mol. Cell. Biol., 12:246-258 ).
  • piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements.
  • the target RNA is mRNA and encodes a peptide or protein.
  • the target RNA to be produced by the in vivo transcription process according to the present invention can preferably encode a wide range of proteins or peptides.
  • the target RNA comprises at least one open reading frame, which encodes a therapeutic protein or peptide, or a fragment, variant or derivative thereof.
  • an antigen is encoded by the at least one open reading frame.
  • the antigen is a pathogenic antigen, a tumor antigen, an allergenic antigen or an autoimmune antigen.
  • the administration of the target RNA encoding the antigen may be used in a genetic vaccination approach against a disease involving said antigen.
  • Fragments or parts of a protein in the context of the present invention are typically understood to be peptides corresponding to a continuous part of the amino acid sequence of a protein, preferably having a length of about 6 to about 20 or even more amino acids, e.g. parts as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence.
  • fragments are typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form.
  • Fragments or parts of the proteins as defined herein may also comprise epitopes or functional sites of those proteins.
  • fragments or parts of a proteins in the context of the invention are antigens, particularly immunogens, e.g. antigen determinants (also called 'epitopes'), or do have antigenic characteristics, eliciting an adaptive immune response. Therefore, fragments of proteins or peptides may comprise at least one epitope of those proteins or peptides.
  • domains of a protein like the extracellular domain, the intracellular domain or the transmembrane domain, and shortened or truncated versions of a protein may be understood to comprise a fragment of a protein.
  • an antibody is encoded by the at least one open reading frame of the target RNA according to the invention.
  • open reading frame may typically be a sequence of several nucleotide triplets, which may be translated into a peptide or protein.
  • An open reading frame preferably starts with a start codon, i.e. a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG or AUG), at its 5'-end and a subsequent region, which usually exhibits a length, which is a multiple of 3 nucleotides.
  • An ORF is preferably terminated by a stop-codon (e.g., TAA, TAG, TGA). Typically, this is the only stop-codon of the open reading frame.
  • an open reading frame in the context of the present invention is preferably a nucleotide sequence, consisting of a number of nucleotides that may be divided by three, which starts with a start codon (e.g. ATG or AUG) and which preferably terminates with a stop codon (e.g., TAA, TGA, or TAG or UAA, UAG, UGA, respectively).
  • the open reading frame may be isolated or it may be incorporated in a longer nucleic acid sequence, for example in a vector or an mRNA.
  • An open reading frame may also be termed 'protein coding region'.
  • the protein that is encoded by the target RNA to be produced by the in vivo transcription process of the present invention is preferably an antigen.
  • antigen refers typically to a substance, which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells.
  • the target RNA according to the present invention may encode a protein or a peptide, which comprises a pathogenic antigen or a fragment, variant or derivative thereof.
  • pathogenic antigens are derived from pathogenic organisms, in particular bacterial, viral or protozoological (multicellular) pathogenic organisms, which evoke an immunological reaction in a subject, in particular a mammalian subject, more particularly a human. More specifically, pathogenic antigens are preferably surface antigens, e.g. proteins (or fragments of proteins, e.g. the exterior portion of a surface antigen) located at the surface of the virus or the bacterial or protozoological organism.
  • surface antigens e.g. proteins (or fragments of proteins, e.g. the exterior portion of a surface antigen
  • Pathogenic antigens are peptide or protein antigens preferably derived from a pathogen associated with infectious disease, which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Bur
  • the target RNA according to the present invention may encode a protein or a peptide, which relates to a tumor antigen.
  • the target RNA according to the present invention may encode a tumor antigen or a fragment, variant or derivative of said tumor antigen selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5- beta-1 -integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1 /m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCAl/m, BRCA2/m, CA 1 5-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CD
  • the target RNA encodes a protein or a peptide, which comprises a therapeutic protein or a fragment, variant or derivative thereof.
  • Therapeutic proteins as defined herein are peptides or proteins, which are beneficial for the treatment of any inherited or acquired disease, or which improve the condition of an individual. Particularly, therapeutic proteins play a big role in the creation of therapeutic agents that could modify and repair genetic errors, destroy cancer cells or pathogen-infected cells, treat immune system disorders, treat metabolic or endocrine disorders, among other functions.
  • the protein hormone erythropoietin (EPO) can be utilized in treating patients with erythrocyte deficiency, which is a common cause of kidney complications.
  • adjuvant proteins and therapeutic antibodies are encompassed by therapeutic proteins as well as hormones used in replacement therapy, which is e.g. used in the therapy of women in menopause.
  • therapeutic proteins may be used for other purposes, e.g. wound healing, tissue regeneration, angiogenesis, etc. Therefore therapeutic proteins can be used for various purposes including treatment of various diseases like e.g. infectious diseases, neoplasms (e.g. cancer or tumor diseases), diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system, independently if they are inherited or acquired.
  • infectious diseases e.g. infectious diseases, neoplasms (e.g. cancer or tumor diseases)
  • diseases of the blood and blood-forming organs endocrine, nutritional and metabolic diseases
  • diseases of the nervous system e.g. infectious diseases, neoplasms (e.g. cancer or tumor diseases)
  • diseases of the blood and blood-forming organs e.g.
  • the present invention is also directed to a vector comprising a DNA sequence encoding the target RNA as defined above and a DNA sequence encoding an RNase inhibitor.
  • the additional DNA sequence encoding an RNase inhibitor will allow producing recombinant RNase inhibitor in the microorganism or host cell producing the target RNA, thereby stabilizing the formed target RNA against degradation.
  • the vector comprises a DNA sequence encoding an RNase inhibitor which is selected from E.coli RNase inhibitors RRaA and RraB and human RNase inhibitor RNH1.
  • the E.coli RNase inhibitors RRaA and RraB are known to inhibit Ribonuclease E (RNase E) of E.coli (see Gao et al., (2006) Mol. Microbiol. 61(2): 394-406 ).
  • Most preferred vectors comprise the DNA sequence encoding human RNase inhibitor RNH1.
  • the present invention is also directed to a microorganism comprising a vector as described above or a first vector comprising a DNA sequence encoding a target RNA and a second vector comprising a DNA sequence encoding an RNase inhibitor.
  • the RNase inhibitor encoded by the vector of the host cell preferably is selected from E.coli RNase inhibitor RraA, E.coli RNase inhibitor RraB and human RNase inhibitor RNH1 and more preferably is human RNase inhibitor RNH1.
  • the present invention is also directed to the use of a microorganism comprising a vector comprising a DNA sequence encoding a target RNA for the in vivo transcription of said DNA sequence into said target RNA anda DNA sequence encoding an RNase inhibitor.
  • the microorganism comprises a first vector comprising a DNA sequence encoding the target RNA and a second vector comprising a DNA sequence encoding an RNase inhibitor.
  • the RNase inhibitor is selected from E.coli RNase inhibitor RraA and human RNase inhibitor RNH1.
  • the present invention is also directed to the use of an expression vector comprising a DNA sequence encoding an RNase inhibitor in a process for the in vivo transcription of a DNA sequence encoding a target RNA.
  • the RNase inhibitor is selected from E.coli RNase inhibitor RraA and human RNase inhibitor RNH1.
  • the cloning of five different vector constructs IB-1, IB-3, IB-4, IB-5, IB-6 encoding the target RNA was carried out as follows: For the target RNA to be transcribed in vivo, a non-coding DNA sequence of 500 base pairs (SEQ ID No. 1) corresponding to the target RNA was inserted into each vector construct. In vector constructs IB-5 and IB-6 the start codon was deleted and three additional stop codons were inserted to prevent any translation into recombinant protein.
  • the DNA sequence encoding the target RNA further included six restriction sites (3 on each end of the sequence (at the 5'-end: BGIII, Ndel, BamHI; at the 3'-end: XhoI, AvrII, BlpI).
  • Vector constructs IB-5 and IB-6 the DNA sequence encoding an RNase inhibitor was additionally inserted.
  • Vector construct IB-5 comprised the DNA sequence of the RraA gene of E.coli strain K12 (Accession No POA8R0; UniProtKB; SEQ ID No. 3)
  • vector construct IB-6 comprised the DNA sequence of the human RNase inhibitor gene RNH1 (Accession No P13489; UniProtKB; SEQ ID No. 4).
  • Vector construct IB-1 (pCV19min-N500-T7-Term) contained a T7 promoter (SEQ ID No. 5), the DNA encoding the non-coding 500 bp target RNA sequence and a T7 terminator (SEQ ID No. 6).
  • Vector construct IB-3 (ptac-RNA500-rrnBT1T2) contained a tac promoter, the DNA sequence encoding the non-coding 500 bp target RNA sequence and a rrnBT1T2 terminator (SEQ ID No. 7).
  • Vector IB-4 (pET-21(+)-RNA500) contained a T7 promoter (SEQ ID No. 5), a lac operator (SEQ ID No. 8), the DNA sequence encoding; the non-coding 500 bp target RNA sequence and a T7 terminator (SEQ ID No. 6).
  • Vector IB-5 (pET-Duet-1-EccraA(Ec)-RNA500) contained a first T7 promoter (SEQ ID No. 5), a first lac operator (SEQ ID No. 8) and the DNA sequence encoding the non-coding, 500 bp target RNA sequence and a second T7 promote r(SEQ ID No. 5), a second lac operator (SEQ ID No. 8) and a DNA sequence encoding RNase inhibitor RraA of E.coli (SEQ ID No. 3)as well as a T7 terminator (SEQ ID No. 6).
  • Vector IB-6 (pET-Duet-1-HsRNH1(Ec)-RNA500) contained a first T7 promoter (SEQ ID No. 5), a first lac operator (SEQ ID No. 8) and the DNA sequence encoding the non-coding, 500 bp target RNA sequence and a second T7 promoter (SEQ ID No. 5), a second lac operator (SEQ ID No. 8) and a DNA sequence encoding the human RNase inhibitor HsRNH1 (SEQ ID No. 4) as well as a T7 terminator (SEQ ID No. 6).
  • the vector constructs IB1-, IB-3, IB-4, IB-5 and IB-6 were subsequently transformed into E.coli strains according to the following Table 1.
  • Table 1 Transformation of E.coli strains wth vector constructs
  • Vector construct Expression system E.coli strain RNase inhibitor Transcript size IB-1 T7 BL21(DE3) - 562 b IB-3 Tac XL10-Gold - 466 b IB-3 Tac SCS110 - 466 b IB-4 T71ac BL21(DE3) - 517 b
  • IB-5 T71ac BL21(DE3)
  • Example 2 In vivo transcription of target RNA
  • the E.coli cultures in 20 ml LB amp - medium were incubated overnight at 37°C and at 142 rpm. From these overnight cultures, 10 ml were used to prepare 50 ml production cultures by adding 40 ml fresh LB amp medium and growing the cultures to OD 600 of 0.6. The cultures were induced by adding 0.5 mM and 1 mM IPTG, respectively, while the temperature was lowered to room temperature. Cultivation was continued for 2h, 4h, 5h, and 20h.
  • RNA from the cultures was subsequently isolated using the RNeasy kit (Qiagen) according to the manufacturer's purification protocol.
  • RNA after in vivo transcription in E.coli was carried out by RT-PCR.
  • the target RNA was converted in a first step into the complementary cDNA.
  • the cDNA was further amplified.
  • the primers used in the RT-PCR and the subsequent PCR had the following sequences.
  • RT-PCR The conditions for RT-PCR were as follows: 0.3 to 1 ⁇ g RNA; 1 ⁇ l primer (15 pmol), water ad 12 ⁇ l; 65°C, 5 min; Subsequently, the following reagents were added: 4 ⁇ l reaction buffer, 1 ⁇ l of 200U/ ⁇ l Revert Aid HMinus M-MulV-RT,1 ⁇ l of Ribolock RNase inhibitor (20U/ ⁇ l) and 2 ⁇ l dNTP mix (10 mM); incubation time was 42°C at 1h followed by 70°C, 5 min to stop the reaction;
  • Conditions for the subsequent PCR were as follows: 2 ⁇ l product from reverse transcription, 25 ⁇ l PCR Master Mix (2x), 5 ⁇ l forward primer (5pmol), 5 ⁇ l reverse primer (5pmol), 1.5 ⁇ l DMSO, 11.5 ⁇ l water;
  • the PCR scheme was as follows: Reaction step Temperature (°C) Time (min:sec) Number of cycles Initial Denaturation 95 2:00 1 Denaturation 95 0:30 20 Annealing 65 0:30 Extension 72 0:30 Completion 72 10:00 1
  • the band at about 355 bp relates to the target RNA to be expected from the in vivo transcribed construct IB-5.
  • Figure 3 clearly shows the signal for the target RNA at 594 bp to be expected for vector construct IB-5 which is not found in the negative control thereby further confirming the formation of the target RNA transcript by the claimed in vivo transcription process.
  • FIG. 4 refers to a sample obtained by in vivo transcription of vector construct IB-5 at 0.5 mM IPTG, 5h.
  • the signal in the middle refers to a sample obtained by in vivo transcription of vector construct IB-5 at 1 mM IPTG, 5h, and the bottom signal refers to a negative control obtained from total RNA of non-transformed E.coli.
  • the samples generated from IB-5 show a signal for 677 bp while this signal is missing in the negative control.
  • electropherograms generated using two different types of instruments allowed the detection and identification of the expected target RNA transcript generated by the in vivo transcription process of the present invention.
  • a positive control (E1) containing in vitro transcribed vector construct IB-1 and total RNA of non-transformed E.coli is shown together with RNA isolated after in vivo transcription of vector construct IB-5 (F1). Both samples were additionally depleted of ribosomal RNA using the Microbe Express kit (Ambion) according to the manufacturer's protocol. Such depletion of ribosomal RNA was not done in the negative control (total RNA of non-transformed E.coli ; D1) in which 16S and 23S RNA consequently still represent the main fractions.
  • the expected band (547 bp) for the in vitro generated RNA transcript is present in the samples of the positive control (see lanes 2 to 7, 9 and 11).
  • Lanes 13 and 14 reveal a band for the in vivo generated target RNA of vector construct IB-5 having a length of 565 bp.
  • At least lane 16 further reveals the in vivo generated target RNA of vector construct IB-5 having a length of 565 bp.
  • the same band is present after depletion of rRNA.
  • a dot blot was carried out using an amount of RNA of 3 ng for each sample.
  • As positive control the in vitro transcribed RNA of vector construct IB-1 was used.
  • Figure 7 shows the result of the dot blot.
EP19216087.7A 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte Pending EP3699288A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP19216087.7A EP3699288A1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP19216087.7A EP3699288A1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte
EP15760098.2A EP3332019B1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte
PCT/EP2015/068310 WO2017025120A1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
EP15760098.2A Division EP3332019B1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte

Publications (1)

Publication Number Publication Date
EP3699288A1 true EP3699288A1 (fr) 2020-08-26

Family

ID=54065318

Family Applications (2)

Application Number Title Priority Date Filing Date
EP15760098.2A Active EP3332019B1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte
EP19216087.7A Pending EP3699288A1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP15760098.2A Active EP3332019B1 (fr) 2015-08-07 2015-08-07 Procédé pour la production in vivo d'arn dans une cellule hôte

Country Status (4)

Country Link
US (1) US20190024096A1 (fr)
EP (2) EP3332019B1 (fr)
DK (1) DK3332019T3 (fr)
WO (1) WO2017025120A1 (fr)

Families Citing this family (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013120497A1 (fr) 2012-02-15 2013-08-22 Curevac Gmbh Acide nucléique comprenant ou codant pour une tige-boucle d'histone et une séquence poly(a) ou un signal de polyadénylation pour l'augmentation de l'expression d'une protéine thérapeutique codée
CA2897858A1 (fr) 2013-02-22 2014-08-28 Curevac Gmbh Combinaison d'une vaccination et de l'inhibition de la voie de pd-1
KR20160044566A (ko) 2013-08-21 2016-04-25 큐어백 아게 호흡기 세포융합 바이러스
ES2931832T3 (es) 2014-06-25 2023-01-03 Acuitas Therapeutics Inc Lípidos y formulaciones de nanopartículas lipídicas novedosos para la entrega de ácidos nucleicos
WO2016091391A1 (fr) 2014-12-12 2016-06-16 Curevac Ag Molécules d'acides nucléiques artificielles destinées à améliorer l'expression de protéines
EP4353257A2 (fr) 2015-04-13 2024-04-17 CureVac Manufacturing GmbH Procédé de production de compositions d'arn
WO2016165831A1 (fr) 2015-04-17 2016-10-20 Curevac Ag Lyophilisation de l'arn
EP3289101B1 (fr) 2015-04-30 2021-06-23 CureVac AG Poly(n)polymérase immobilisée
EP3294885B1 (fr) 2015-05-08 2020-07-01 CureVac Real Estate GmbH Procédé de production d'arn
CN107810009A (zh) 2015-05-15 2018-03-16 库瑞瓦格股份公司 涉及施用至少一种mRNA构建体的初免‑加强方案
CN107635587A (zh) 2015-05-20 2018-01-26 库瑞瓦格股份公司 包含长链rna的干燥粉末组合物
EP3297682B1 (fr) 2015-05-20 2021-07-14 CureVac AG Composition de poudre sèche comprenant de l'arn à chaîne longue
DK3303583T3 (da) 2015-05-29 2020-07-13 Curevac Real Estate Gmbh Fremgangsmåde til fremstilling og oprensning af rna, omfattende mindst et trin til tangential-flow-filtrering
US11608513B2 (en) 2015-05-29 2023-03-21 CureVac SE Method for adding cap structures to RNA using immobilized enzymes
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US10501768B2 (en) 2015-07-13 2019-12-10 Curevac Ag Method of producing RNA from circular DNA and corresponding template DNA
EP3362576A1 (fr) 2015-10-12 2018-08-22 CureVac AG Procédé automatisé d'isolement, de sélection et/ou de détection de micro-organismes ou de cellules présents dans une solution
HUE061564T2 (hu) 2015-10-28 2023-07-28 Acuitas Therapeutics Inc Új lipidek és lipid nanorészecske készítmények nukleinsavak bevitelére
US11413346B2 (en) 2015-11-09 2022-08-16 Curevac Ag Rotavirus vaccines
KR20180107109A (ko) 2015-12-22 2018-10-01 큐어백 아게 Rna 분자 조성물의 제조 방법
WO2017109161A1 (fr) 2015-12-23 2017-06-29 Curevac Ag Procédé de transcription in vitro d'arn utilisant un tampon contenant un acide dicarboxyliqlue ou un acide tricarboxylique ou un sel de celui-ci
MX2018009917A (es) 2016-02-17 2019-08-14 Curevac Ag Vacuna contra el virus del zika.
US11920174B2 (en) 2016-03-03 2024-03-05 CureVac SE RNA analysis by total hydrolysis and quantification of released nucleosides
EP3448427A1 (fr) 2016-04-29 2019-03-06 CureVac AG Arn codant pour un anticorps
EP3452101A2 (fr) 2016-05-04 2019-03-13 CureVac AG Arn codant pour une protéine thérapeutique
WO2017191264A1 (fr) 2016-05-04 2017-11-09 Curevac Ag Molécules d'acide nucléique et leurs utilisations
SG11201809912UA (en) 2016-06-09 2018-12-28 Curevac Ag Hybrid carriers for nucleic acid cargo
WO2018096179A1 (fr) 2016-11-28 2018-05-31 Curevac Ag Procédé de purification d'arn
US11464836B2 (en) 2016-12-08 2022-10-11 Curevac Ag RNA for treatment or prophylaxis of a liver disease
WO2018104540A1 (fr) 2016-12-08 2018-06-14 Curevac Ag Arn pour la cicatrisation des plaies
WO2018115507A2 (fr) 2016-12-23 2018-06-28 Curevac Ag Vaccin contre l'hénipavirus
EP3558356A2 (fr) 2016-12-23 2019-10-30 CureVac AG Vaccin contre le coronavirus du syndrome respiratoire du moyen-orient
EP3558354A1 (fr) 2016-12-23 2019-10-30 CureVac AG Vaccin contre le virus de lassa
WO2018161053A1 (fr) * 2017-03-02 2018-09-07 MEIRAGTX, UK II Limited Régulation de l'expression génique par clivage par la rnase p modulé par aptamère
EP3601576A1 (fr) 2017-03-24 2020-02-05 CureVac AG Acides nucléiques codant pour des protéines associées à crispr et leurs utilisations
WO2018179834A1 (fr) * 2017-03-28 2018-10-04 Ajinomoto Co., Inc. Procédé de production d'arn
WO2018191657A1 (fr) 2017-04-13 2018-10-18 Acuitas Therapeutics, Inc. Lipides pour administration d'agents actifs
AU2018256877B2 (en) 2017-04-28 2022-06-02 Acuitas Therapeutics, Inc. Novel carbonyl lipids and lipid nanoparticle formulations for delivery of nucleic acids
CN111328287A (zh) 2017-07-04 2020-06-23 库瑞瓦格股份公司 新型核酸分子
EP3668833A1 (fr) 2017-08-16 2020-06-24 Acuitas Therapeutics, Inc. Lipides destinés à être utilisés dans des formulations nanoparticulaires lipidiques
WO2019036030A1 (fr) 2017-08-17 2019-02-21 Acuitas Therapeutics, Inc. Lipides destinés à être utilisés dans des formulations de nanoparticules lipidiques
US11542225B2 (en) 2017-08-17 2023-01-03 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
EP3673069A1 (fr) 2017-08-22 2020-07-01 CureVac AG Vaccin contre les bunyavirus
SG11202003247RA (en) 2017-11-08 2020-05-28 Curevac Ag Rna sequence adaptation
EP3723796A1 (fr) 2017-12-13 2020-10-21 CureVac AG Vaccin contre les flavivirus
EP3728634A1 (fr) 2017-12-21 2020-10-28 CureVac AG Adn linéaire double brin couplé à un support ou une étiquette unique et procédés de production dudit adn linéaire double brin
US11241493B2 (en) 2020-02-04 2022-02-08 Curevac Ag Coronavirus vaccine
US11576966B2 (en) 2020-02-04 2023-02-14 CureVac SE Coronavirus vaccine
JP2024502210A (ja) 2020-12-22 2024-01-17 キュアバック エスイー ＳＡＲＳ－ＣｏＶ－２バリアントに対するＲＮＡワクチン

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024904A2 (fr) 2000-09-23 2002-03-28 Bachmann Till T Procede de production d'acide ribonucleique (arn)
US6630306B1 (en) 1996-12-19 2003-10-07 Yale University Bioreactive allosteric polynucleotides
WO2007144508A2 (fr) * 2006-06-14 2007-12-21 Universite Rene Descartes - Paris V Arn de transfert chimerique et son utilisation pour la production d'arn par une cellule
WO2012006732A9 (fr) 2010-07-16 2012-02-23 Universite De Montreal Purification par affinité d'arn dans des conditions d'origine sur la base de l'interaction lambda boîteb/n peptide
US8143061B2 (en) 2003-07-23 2012-03-27 Centre National De La Recherche Scientifique Industrial method for producing RNA and system for carrying out said method
WO2014140211A1 (fr) 2013-03-15 2014-09-18 Novartis Ag Procédés de purification de l'arn
WO2015024667A1 (fr) 2013-08-21 2015-02-26 Curevac Gmbh Procédé pour augmenter l'expression de protéines codées par l'arn

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6630306B1 (en) 1996-12-19 2003-10-07 Yale University Bioreactive allosteric polynucleotides
WO2002024904A2 (fr) 2000-09-23 2002-03-28 Bachmann Till T Procede de production d'acide ribonucleique (arn)
US8143061B2 (en) 2003-07-23 2012-03-27 Centre National De La Recherche Scientifique Industrial method for producing RNA and system for carrying out said method
WO2007144508A2 (fr) * 2006-06-14 2007-12-21 Universite Rene Descartes - Paris V Arn de transfert chimerique et son utilisation pour la production d'arn par une cellule
WO2012006732A9 (fr) 2010-07-16 2012-02-23 Universite De Montreal Purification par affinité d'arn dans des conditions d'origine sur la base de l'interaction lambda boîteb/n peptide
WO2014140211A1 (fr) 2013-03-15 2014-09-18 Novartis Ag Procédés de purification de l'arn
WO2015024667A1 (fr) 2013-08-21 2015-02-26 Curevac Gmbh Procédé pour augmenter l'expression de protéines codées par l'arn

Non-Patent Citations (35)

* Cited by examiner, † Cited by third party
Title
"GeneBank", Database accession no. X65327
"UniProtKB", Database accession no. P13489
"UniProtKB", Database accession no. POA8RO
CHATTOPADHYAY ET AL., PROC. NATL. ACAD. SCI., vol. 92, 1995, pages 4061 - 4065
CHENELINGTON, PLOS COMPUT BIOL, vol. 5, no. 12, 2009, pages elO00620
CILLEYWILLIAMSON, RNA, vol. 9, 2003, pages 663 - 676
FABER ET AL., J. BIOL. CHEM., vol. 276, no. 34, pages 32064 - 32070
G. DI TOMASSO ET AL: "The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions", NUCLEIC ACIDS RESEARCH, vol. 39, no. 3, E18, 11 November 2010 (2010-11-11), pages 1 - 10, XP055082907, ISSN: 0305-1048, DOI: 10.1093/nar/gkq1084 *
GAO ET AL., MOL. MICROBIOL., vol. 61, no. 2, 2006, pages 394 - 406
GRATEWILSON, PROC. NAT. ACAD. SCI, vol. 96, 1999, pages 6131 - 6136
HERMANSON, BIOCONJUGATE TECHNIQUES, 1996
JUOZAS SIURKUS ET AL: "Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression", MICROBIAL CELL FACTORIES, vol. 10, no. 1, 65, 8 August 2011 (2011-08-08), BIOMED CENTRAL, LONDON, NL, pages 1 - 11, XP021105373, ISSN: 1475-2859, DOI: 10.1186/1475-2859-10-65 *
KIM ET AL., GENES DEV., vol. 22, 2008, pages 3497 - 3508
KOIZUMI ET AL., NATURE STRUCT. BIOL., vol. 6, no. 11, 1999, pages 1062 - 1071
LEE ET AL., CELL, vol. 114, no. 5, 2003, pages 623 - 634
LIU YAMEI ET AL: "DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.", BMC BIOTECHNOLOGY, vol. 10, 85, 2010, pages 1 - 9, XP002746283, ISSN: 1472-6750 *
NEELYFRIEDMAN, MOLECULAR MICROBIOLOGY, vol. 38, no. 5, 2000, pages 1074 - 1085
NELISSEN ET AL., NUCL. ACIDS RES., 2012, pages 1 - 12
NELISSEN FRANK H T ET AL: "Fast production of homogeneous recombinant RNA--towards large-scale production of RNA.", NUCLEIC ACIDS RESEARCH, vol. 40, no. 13, E102, July 2012 (2012-07-01), pages 1 - 12, XP002746282, ISSN: 1362-4962 *
PONCHON LUC ET AL: "A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold.", NATURE PROTOCOLS 2009, vol. 4, no. 6, 2009, pages 947 - 959, XP002747452, ISSN: 1750-2799 *
QI ET AL., BIOCHEMISTRY, vol. 49, 2010, pages 5782 - 5789
ROTH, A. ET AL., RNA, vol. 12, 2006, pages 607 - 619
ROTHBREAKER, ANNU REV BIOCHEM, vol. 78, 2009, pages 305 - 334
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHUBERTKURRECK, CURR. DRUG TARGETS, vol. 5, no. 8, 2004, pages 667 - 681
SEETHERMAN ET AL., NATURE BIOTECH., vol. 19, 2001, pages 336 - 341
SETO ET AL., MOLECULAR CELL, vol. 26, no. 5, 2007, pages 603 - 609
SHIH ET AL., ANNUAL REVIEW OF BIOCHEMISTRY, vol. 71, 2002, pages 887 - 917
SIOMI ET AL., NAT. REV. MOL. CELL. BIOL., vol. 12, 2011, pages 246 - 258
SRISAWAT ET AL., NUCL. ACIDS RES., vol. 29, no. 2, 2001, pages e4
SRISAWATENGELKE, RNA, vol. 7, 2001, pages 632 - 641
SU ET AL., BIOCHEMISTRY, vol. 36, 1997, pages 12722 - 12732
TAVERNIER ET AL., J CONTROL RELEASE, vol. 150, no. 3, 2011, pages 238 - 47
WINKLER ET AL., NATURE, vol. 428, 2004, pages 281 - 286
YEOM JI-HYUN ET AL: "Inhibitory effects of RraA and RraB on RNAse E-related enzymes imply conserved functions in the regulated enzymatic cleavage of RNA.", FEMS MICROBIOLOGY LETTERS, vol. 285, no. 1, August 2008 (2008-08-01), pages 10 - 15, XP002746281, ISSN: 0378-1097 *

Also Published As

Publication number Publication date
WO2017025120A1 (fr) 2017-02-16
DK3332019T3 (da) 2020-02-17
EP3332019B1 (fr) 2019-12-18
EP3332019A1 (fr) 2018-06-13
US20190024096A1 (en) 2019-01-24

Similar Documents

Publication Publication Date Title
EP3332019B1 (fr) Procédé pour la production in vivo d'arn dans une cellule hôte
JP7245878B2 (ja) 人工核酸分子
JP6997251B2 (ja) 人工核酸分子
JP6907116B2 (ja) 人工核酸分子
EP3317424B1 (fr) Procédé d'analyse d'une molécule d'arn
AU2023222981A1 (en) Materials and methods for engineering cells and uses thereof in immuno-oncology
JP6772062B2 (ja) 癌の免疫療法
WO2019092153A1 (fr) Adaptation de séquence d'arn
US11879145B2 (en) Reagents and methods for replication, transcription, and translation in semi-synthetic organisms
EP3983539A1 (fr) Arn circulaires pour thérapie cellulaire
WO2019036513A1 (fr) Acides nucléiques stabilisés codant pour de l'acide ribonucléique messager (arnm)
US10350307B2 (en) In vivo RNA or protein expression using double-stranded concatemeric DNA including phosphorothioated nucleotides
WO2022198080A1 (fr) Édition multiplex avec des enzymes cas
US20200362382A1 (en) Methods of preparing modified rna
KR20240035752A (ko) 원형 폴리리보뉴클레오티드의 농축 방법
WO2023201294A1 (fr) Variants d'arn polymérase
KR20220055399A (ko) 세포질에서 자가 전사가 가능한 mRNA를 제공하는 RNA/DNA 시스템

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20191213

AC Divisional application: reference to earlier application

Ref document number: 3332019

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR